Molecular Biochemical Study on the Cyanobacterial ndh (NADH dehydrogenase) Gene Products

蓝藻ndh（NADH脱氢酶）基因产物的分子生化研究

• 批准号: 06640840
• 负责人: TAKAHASHI Yasuhiro
• 金额: $ 1.41万
• 依托单位: Osaka University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1994
• 资助国家: 日本
• 起止时间: 1994 至 1995
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-06640840/
• 关键词: Cyanobacteria; ndh genes; NAD (P) H dehydrogenase; NAD (P) H脱水素酵素; NAD(P)H 脱水素酵素

Eleven ndh genes (ndhA-K) have been identified in chloroplast and cyanobacterial genomes on the basis of sequence similarity to the subunits of mitochondrial and bacterial NADH : ubiquinone oxidoreductase. These findings have been taken to indicate the presence of an enzyme related to the mitochondrial and bacterial enzymes, possibly an NAD (P) H : plastoquinone oxidoreductase ; however, the active enzyme complex has not yet been isolated. In order to clarify its physiological significance, several attempts have been made using the filamentous cyanobacterium Plectonema boryanum, a facultative chemoheterotroph.Three essential ndh genes are missing from the plastid genomes. They are 51 (FP), 24 (FP), and 75 (IP) subunits of bovine complex I,which constitute a subcomplex involved in the first part of electron pathway from NADH to intermediate acceptors. Cyanobacterial DNA fragment was cloned after PCR amplification, which shows sequence similarity to the mitochondrial 51 (FP) subunit. Attempts to inactivate the ndhA,ndhI,ndhG,or ndhE gene of P.boryanum have been unsuccessful under both normal and high-CO2 growth conditions, suggesting an essential role in this organism. In order to identify ndh related genes, we have developed a system for mutagenesis using a non-replicating plasmid pTAF10 which was constructed from pUC12, the neo gene, and OMEGA fragment. Genomic fragments partially digested by Sau3A were ligated into pTAF10, introduced into the cells by electroporation and kanamycin resistant mutants were obtained after single cross-over recombination. Insertion of the entire plasmid into the chromosome may interrupt the gene or disturb the expression of flanking genes due to the terminator sequence in the OMEGA fragment. Screening for the defect in chemoheterotrophic growth resulted in the isolation of dozens of mutants which are now analyzed.

已经在叶绿体和蓝细菌基因组中鉴定了11个NDH基因（NDHA-K），基于与线粒体和细菌NADH的亚基的序列相似性：泛素酮氧化还原酶。这些发现已被认为表明存在与线粒体和细菌酶有关的酶，可能是NAD（P）H：plasto酮氧化还原酶。但是，活性酶复合物尚未分离。为了阐明其生理意义，使用丝状蓝细菌boryanum进行了几次尝试，这是一种兼性化学果蝇。它们是牛I的51（FP），24（FP）和75（IP）亚基I，它们构成了从NADH到中间受体的电子途径的第一部分的子复合物。 PCR扩增后克隆蓝细菌DNA片段，该片段显示与线粒体51（FP）亚基的序列相似性。在正常和高CO2生长条件下，试图使P.Boryanum的NDHA，NDHI，NDHG或NDHE基因失败，这表明在该生物体中起着至关重要的作用。为了识别NDH相关的基因，我们使用了由PUC12，NEO基因和欧米茄片段构建的非复制质粒PTAF10开发了诱变系统。将SAU3A部分消化的基因组片段连接到PTAF10中，通过电穿孔引入细胞，单次交叉重组后获得了抗Kanamycin抗性突变体。将整个质粒插入染色体中可能会中断基因或由于欧米茄片段中的终结序列而导致侧翼基因的表达。筛查化学嗜营养物质生长的缺陷导致分析了数十个突变体，这些突变体现已被分析。

项目成果

Hiroshi Matsubara: "Three Iron-Sulfur Proteins Encoded by Three ORFs in Chloroplasts and Cyanobacteria." Photosyn. Res.46. 107-115 (1995)

Hiroshi Matsubara：“叶绿体和蓝藻中三个 ORF 编码的三种铁硫蛋白。”

南善子: "広く生体反応に関わる鉄硫黄蛋白質" 生物物理. 35. 7-12 (1995)

Yoshiko Minami：“广泛参与生物反应的铁硫蛋白”生物物理学 35. 7-12 (1995)。

Hiroshi Matsubara, Hirozo Oh-oka, Yasuhiro Takahashi, Yuichi Fujita: "Three Iron-Sulfur Proteins Encoded by Three ORFs in Chloroplasts and Cyanobacteria." Photosyn.Res.46 (1-2). 107-115 (1995)

Hiroshi Matsubara、Hirozo Oh-​​oka、Yasuhiro Takahashi、Yuichi Fujita：“叶绿体和蓝藻中三个 ORF 编码的三种铁硫蛋白。”

Yasuhiro Takahashi, Takako Michii, Tetuyuki Akashi, Yuichi Fujita: "Cyanobacterial Mutagenesis Mediated by OMEGA-Containing Plasmid." (in preparation).

Yasuhiro Takahashi、Takako Michii、Tetuyuki Akashi、Yuichi Fujita：“含 OMEGA 的质粒介导的蓝藻诱变。”

Yoshiko Minami, Yasuhiro Takahashi, Hiroshi Matsubara: "Iron-Sulfur Proteins Involved in Various Biological Reactions." Biophysics. 35 (2). 7-12 (1995)

Yoshiko Minami、Yasuhiro Takahashi、Hiroshi Matsubara：“参与各种生物反应的铁硫蛋白。”